Recombinant vwf formulations

ABSTRACT

The present invention provides long-term stable pharmaceutical formulations of recombinant von-Willebrand Factor (rVWF) and methods for making and administering said formulations.

This application claims priority of U.S. Provisional Application No.61/017,418, filed Dec. 28, 2007, and U.S. Provisional Application No.61/017,881 filed Dec. 31, 2007, each of which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

Generally, the invention relates to formulations of recombinant VWF andmethods for making a composition comprising recombinant VWF.

BACKGROUND OF THE INVENTION

Von Willebrand factor (VWF) is a glycoprotein circulating in plasma as aseries of multimers ranging in size from about 500 to 20,000 kD.Multimeric forms of VWF are composed of 250 kD polypeptide subunitslinked together by disulfide bonds. VWF mediates initial plateletadhesion to the sub-endothelium of the damaged vessel wall. Only thelarger multimers exhibit hemostatic activity. It is assumed thatendothelial cells secrete large polymeric forms of VWF and those formsof VWF which have a low molecular weight (low molecular weight VWF)arise from proteolytic cleavage. The multimers having large molecularmasses are stored in the Weibel-Pallade bodies of endothelial cells andliberated upon stimulation.

VWF is synthesized by endothelial cells and megakaryocytes as prepro-VWFthat consists to a large extent of repeated domains. Upon cleavage ofthe signal peptide, pro-VWF dimerizes through disulfide linkages at itsC-terminal region. The dimers serve as protomers for multimerization,which is governed by disulfide linkages between the free end termini.The assembly to multimers is followed by the proteolytic removal of thepropeptide sequence (Leyte et al., Biochem. J. 274 (1991), 257-261).

The primary translation product predicted from the cloned cDNA of VWF isa 2813-residue precursor polypeptide (prepro-VWF). The prepro-VWFconsists of a 22 amino acid signal peptide and a 741 amino acidpropeptide, with the mature VWF comprising 2050 amino acids (Ruggeri Z.A., and Ware, J., FASEB J., 308-316 (1993)).

Defects in VWF are causal to Von Willebrand disease (VWD), which ischaracterized by a more or less pronounced bleeding phenotype. VWD type3 is the most severe form in which VWF is completely missing, and VWDtype 1 relates to a quantitative loss of VWF and its phenotype can bevery mild. VWD type 2 relates to qualitative defects of VWF and can beas severe as VWD type 3. VWD type 2 has many sub forms, some beingassociated with the loss or the decrease of high molecular weightmultimers. Von Willebrand syndrome type 2a (VWS-2A) is characterized bya loss of both intermediate and large multimers. VWS-2B is characterizedby a loss of highest-molecular-weight multimers. Other diseases anddisorders related to VWF are known in the art.

US. Pat. Nos. 6,531,577, 7,166,709, and European Patent Application No.04380188.5, describe plasma-derived VWF formulations. However, inaddition to quantity and purity issues with plasma-derived VWF, there isalso a risk of blood-born pathogens (e.g., viruses and VariantCreutzfeldt-Jakob disease (vCJD).

Thus there exists a need in the art to develop a stable pharmaceuticalformulation comprising recombinant VWF.

SUMMARY OF THE INVENTION

The present invention provides formulations useful for compositionscomprising recombinant VWF, resulting in a highly stable pharmaceuticalcomposition. The stable pharmaceutical composition is useful as atherapeutic agent in the treatment of individuals suffering fromdisorders or conditions that can benefit from the administration ofrecombinant VWF.

In one embodiment, the invention provides a stable liquid pharmaceuticalformulation of a recombinant von Willebrand Factor (rVWF) comprising:(a) a rVWF; (b) a buffering agent; (c) one or more salts; (d) optionallya stabilizing agent; and (e) optionally a surfactant; wherein the rVWFcomprises a polypeptide selected from the group consisting of: a) theamino acid sequence set out in SEQ ID NO: 3; b) a biologically activeanalog, fragment or variant of a); c) a polypeptide encoded by thepolynucleotide set out in SEQ ID NO: 1; d) a biologically active analog,fragment or variant of c); and e) a polypeptide encoded by apolynucleotide that hybridizes to the polynucleotide set out in SEQ IDNO: 1 under moderately stringent hybridization conditions; wherein thebuffer is comprised of a pH buffering agent in a range of about 0.1 mMto about 500 mM and wherein the pH is in a range of about 2.0 to about12.0; wherein the salt is at a concentration of about 1 to 500 mM;wherein the stabilizing agent is at a concentration of about 0.1 to 1000mM; and wherein the surfactant is at a concentration of about 0.01 g/Lto 0.5 g/L.

In another embodiment, the aforementioned formulation is providedwherein the rVWF comprises the amino acid sequence set out in SEQ ID NO:3. In another embodiment, an aforementioned formulation is providedwherein the buffering agent is selected from the group consisting ofsodium citrate, glycine, histidine, Tris and combinations of theseagents. In yet another embodiment, an aforementioned formulation isprovided wherein the buffering agent is citrate. In still anotherembodiment of the invention, the aforementioned formulation is providedwherein pH is in the range of 6.0-8.0, or 6.5-7.3. In a relatedembodiment, the aforementioned formulation is provided wherein the pH is7.0. In another embodiment, an aforementioned formulation is providedwherein the buffering agent is citrate and the pH is 7.0.

In still another embodiment, an aforementioned formulation is providedwherein the salt is selected from the group consisting of calciumchloride, sodium chloride and magnesium chloride. In another embodiment,the aforementioned formulation is provided wherein the salt is at aconcentration range of 0.5 to 300 mM. In another embodiment, theaforementioned formulation is provided wherein the salt is calciumchloride at a concentration of 10 mM.

In another embodiment, an aforementioned formulation is provided whereinthe rVWF comprises the amino acid sequence set out in SEQ ID NO: 3;wherein the buffering agent is citrate and the pH is 7.0; and whereinthe salt is calcium chloride at a concentration of 10 mM. In stillanother embodiment, an aforementioned formulation is provided whereinthe rVWF comprises the amino acid sequence set out in SEQ ID NO: 3;wherein the buffering agent is sodium citrate and the pH is 7.0; andwherein the salt is calcium chloride at a concentration of 10 mM andNaCl at a concentration of 100 mM.

Other formulations are also contemplated by the instant invention. Forexample, in one embodiment, an aforementioned formulation is providedwherein the one or more buffering agents is histidine and Tris at aconcentration of 3.3 mM each. In another embodiment, the aforementionedformulation is provided wherein the pH is 7.0. In yet anotherembodiment, an aforementioned formulation is provided wherein the firstsalt is sodium chloride at a concentration of 30 mM and the second saltis calcium chloride at a concentration of 0.56 mM.

In still another embodiment of the invention, an aforementionedformulation is provided wherein the stabilizing agent is selected fromthe group consisting of mannitol, lactose, sorbitol, xylitol, sucrose,trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose,gentiobiose, isomaltose, arabinose, glucosamine, fructose andcombinations of these stabilizing agents. In another embodiment, theaforementioned formulation is provided wherein the stabilizing agentsare trehalose at a concentration of 7.8 mM and mannitol at aconcentration of 58.6 mM.

In another embodiment, an aforementioned formulation is provided whereinthe surfactant is selected from the group consisting of digitonin,Triton X-100, Triton X-114, TWEEN-20, TWEEN-80 and combinations of thesesurfactants. In another embodiment, the aforementioned formulation isprovided wherein the surfactant is TWEEN-80 at 0.03 g/L.

In one embodiment of the invention, an aforementioned formulation isprovided wherein the rVWF comprises amino acid sequence set out in SEQID NO: 3; wherein the buffering agents are histidine at a concentrationof 3.3 mM and Tris at a concentration of 3.3 mM at pH 7.0; wherein thefirst salt is sodium chloride at a concentration of 30 mM and the secondsalt is calcium chloride at a concentration of 0.56 mM; wherein thestabilizing agents are trehalose at a concentration of 7.8 mM mannitolat a concentration of 58.6 mM; and wherein the surfactant is TWEEN-80 at0.03 g/L.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that rVWF is not stable in Advate buffer after 26 weeks,due to the presence of glutathione.

FIG. 2 shows that rVWF is stable in Advate 1:3 buffer for up to 12 weeksat 4° C.

FIG. 3 shows that the stability of a citrate-based formulation is betterthan Advate 1:3 buffer formulation containing 0.1M glutathione.

FIG. 4 shows that rVWF concentration is stable over 26 weeks in Advatebuffer.

FIG. 5 shows that rVWF concentration is stable over time in Advate 1:3buffer.

FIG. 6 shows that rVWF concentration is stable over time incitrate-based buffer.

FIG. 7 shows that most excipients increase the unfolding temperature ofrVWF by about 1 or 2° C.

FIG. 8 shows that 10 mM CaCl₂ increases unfolding temperature of rVWF byabout 8° C. to about 67° C.

FIG. 9 shows that the effect of CaCl₂ is similar at pH 7.3 and pH 6.5.

DETAILED DESCRIPTION OF THE INVENTION Definition of Terms

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Singleton, et al., DICTIONARY OF MICROBIOLOGY ANDMOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger, et al. (eds.), Springer Verlag (1991); and Hale and Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991).

Each publication, patent application, patent, and other reference citedherein is incorporated by reference in its entirety to the extent thatit is not inconsistent with the present disclosure.

It is noted here that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “comprising,” with respect to a peptide compound, means that acompound may include additional amino acids at either or both amino andcarboxy termini of the given sequence. Of course, these additional aminoacids should not significantly interfere with the activity of thecompound. With respect to a composition of the instant invention, theterm “comprising” means that a composition may include additionalcomponents. These additional components should not significantlyinterfere with the activity of the composition.

The term “pharmacologically active” means that a substance so describedis determined to have activity that affects a medical parameter (e.g.,but not limited to blood pressure, blood cell-count, cholesterol level)or disease state (e.g., but not limited to cancer, autoimmunedisorders).

As used herein the terms “express,” “expressing” and “expression” meanallowing or causing the information in a gene or DNA sequence to becomemanifest, for example, producing a protein by activating the cellularfunctions involved in transcription and translation of a correspondinggene or DNA sequence. A DNA sequence is expressed in or by a cell toform an “expression product” such as a protein. The expression productitself, e.g. the resulting protein, may also be said to be “expressed.”An expression product can be characterized as intracellular,extracellular or secreted. The term “intracellular” means inside a cell.The term “extracellular” means outside a cell, such as a transmembraneprotein. A substance is “secreted” by a cell if it appears insignificant measure outside the cell, from somewhere on or inside thecell.

As used herein a “polypeptide” refers to a polymer composed of aminoacid residues, structural variants, related naturally-occurringstructural variants, and synthetic non-naturally occurring analogsthereof linked via peptide bonds. Synthetic polypeptides can beprepared, for example, using an automated polypeptide synthesizer. Theterm “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides.

As used herein a “fragment” of a polypeptide is meant to refer to anyportion of a polypeptide or protein smaller than the full-lengthpolypeptide or protein expression product.

As used herein an “analog” refers to any of two or more polypeptidessubstantially similar in structure and having the same biologicalactivity, but can have varying degrees of activity, to either the entiremolecule, or to a fragment thereof. Analogs differ in the composition oftheir amino acid sequences based on one or more mutations involvingsubstitution of one or more amino acids for other amino acids.Substitutions can be conservative or non-conservative based on thephysico-chemical or functional relatedness of the amino acid that isbeing replaced and the amino acid replacing it.

As used herein a “variant” refers to a polypeptide, protein or analogthereof that is modified to comprise additional chemical moieties notnormally a part of the molecule. Such moieties may modulate themolecule's solubility, absorption, biological half-life, etc. Themoieties may alternatively decrease the toxicity of the molecule andeliminate or attenuate any undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed in Remington'sPharmaceutical Sciences (1980). Procedure for coupling such moieties toa molecule are well known in the art. For example, the variant may be ablood clotting factor having a chemical modification which confers alonger half-life in vivo to the protein. In various aspects,polypeptides are modified by glycosylation, pegylation, and/orpolysialylation.

Recombinant VWF

The polynucleotide and amino acid sequences of prepro-VWF are set out inSEQ ID NO:1 and SEQ ID NO:2, respectively, and are available at GenBankAccession Nos. NM_(—)000552 and NP_(—)000543, respectively. The aminoacid sequence corresponding to the mature VWF protein is set out in SEQID NO: 3 (corresponding to amino acids 764-2813 of the full lengthprepro-VWF amino acid sequence).

One form of useful rVWF has at least the property of invivo-stabilizing, e.g. binding, of at least one Factor VIII (FVIII)molecule and having optionally a glycosylation pattern which ispharmacologically acceptable. Specific examples thereof include VWFwithout A2 domain thus resistant to proteolysis (Lankhof et al., Thromb.Haemost. 77: 1008-1013, 1997), and the VWF fragment from Val 449 to Asn730 including the glycoprotein lb-binding domain and binding sites forcollagen and heparin (Pietu et al., Biochem. Biophys. Res. Commun. 164:1339-1347, 1989). The determination of the ability of a VWF to stabilizeat least one FVIII molecule can be carried out in VWF-deficient mammalsaccording to methods known in the state in the art.

The rVWF of the present invention may be produced by any method known inthe art. One specific example is disclosed in WO86/06096 published onOct. 23, 1986 and U.S. patent application Ser. No. 07/559,509, filed onJul. 23, 1990, which is incorporated herein by reference with respect tothe methods of producing recombinant VWF. Thus, methods are known in theart for (i) the production of recombinant DNA by genetic engineering,e.g. via reverse transcription of RNA and/or amplification of DNA, (ii)introducing recombinant DNA into procaryotic or eucaryotic cells bytransfection, e.g. via electroporation or microinjection, (iii)cultivating said transformed cells, e.g. in a continuous or batchwisemanner, (iv) expressing VWF, e.g. constitutively or upon induction, and(v) isolating said VWF, e.g. from the culture medium or by harvestingthe transformed cells, in order to (vi) obtain purified rVWF, e.g. viaanion exchange chromatography or affinity chromatography. A recombinantVWF may be made in transformed host cells using recombinant DNAtechniques well known in the art. For instance, sequences coding for thepolypeptide could be excised from DNA using suitable restrictionenzymes.

Alternatively, the DNA molecule could be synthesized using chemicalsynthesis techniques, such as the phosphoramidate method. Also, acombination of these techniques could be used.

The invention also provides vectors encoding polypeptides of theinvention in an appropriate host. The vector comprises thepolynucleotide that encodes the polypeptide operatively linked toappropriate expression control sequences. Methods of effecting thisoperative linking, either before or after the polynucleotide is insertedinto the vector, are well known. Expression control sequences includepromoters, activators, enhancers, operators, ribosomal binding sites,start signals, stop signals, cap signals, polyadenylation signals, andother signals involved with the control of transcription or translation.The resulting vector having the polynucleotide therein is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art, including, forexample, compatibility with the chosen expression vector, toxicity ofthe peptides encoded by the DNA molecule, rate of transformation, easeof recovery of the peptides, expression characteristics, bio-safety andcosts. A balance of these factors must be struck with the understandingthat not all host cells are equally effective for the expression of aparticular DNA sequence. Within these general guidelines, usefulmicrobial host cells include bacteria, yeast and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the polypeptides are purified from culture by methodswell known in the art.

Depending on the host cell utilized to express a compound of theinvention, carbohydrate (oligosaccharide) groups may conveniently beattached to sites that are known to be glycosylation sites in proteins.Generally, O-linked oligosaccharides are attached to serine (Ser) orthreonine (Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids not countingproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Alternatively, the compounds may be made by synthetic methods. Forexample, solid phase synthesis techniques may be used. Suitabletechniques are well known in the art, and include those described inMerrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis andPanayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis etal. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), SolidPhase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976),The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), TheProteins (3rd ed.) 2: 257-527. Solid phase synthesis is the preferredtechnique of making individual peptides since it is the mostcost-effective method of making small peptides.

Fragments, Variants and Analogs of VWF

Methods for preparing polypeptide fragments, variants or analogs arewell-known in the art.

Fragments of a polypeptide are prepared using, without limitation,enzymatic cleavage (e.g., trypsin, chymotrypsin) and also usingrecombinant means to generate a polypeptide fragments having a specificamino acid sequence. Polypeptide fragments may be generated comprising aregion of the protein having a particular activity, such as amultimerization domain or any other identifiable VWF domain known in theart.

Methods of making polypeptide analogs are also well-known. Amino acidsequence analogs of a polypeptide can be substitutional, insertional,addition or deletion analogs. Deletion analogs, including fragments of apolypeptide, lack one or more residues of the native protein which arenot essential for function or immunogenic activity. Insertional analogsinvolve the addition of, e.g., amino acid(s) at a non-terminal point inthe polypeptide. This analog may include insertion of an immunoreactiveepitope or simply a single residue. Addition analogs, includingfragments of a polypeptide, include the addition of one or more aminoacids at either of both termini of a protein and include, for example,fusion proteins.

Substitutional analogs typically exchange one amino acid of thewild-type for another at one or more sites within the protein, and maybe designed to modulate one or more properties of the polypeptidewithout the loss of other functions or properties. In one aspect,substitutions are conservative substitutions. By “conservative aminoacid substitution” is meant substitution of an amino acid with an aminoacid having a side chain of a similar chemical character. Similar aminoacids for making conservative substitutions include those having anacidic side chain (glutamic acid, aspartic acid); a basic side chain(arginine, lysine, histidine); a polar amide side chain (glutamine,asparagine); a hydrophobic, aliphatic side chain (leucine, isoleucine,valine, alanine, glycine); an aromatic side chain (phenylalanine,tryptophan, tyrosine); a small side chain (glycine, alanine, serine,threonine, methionine); or an aliphatic hydroxyl side chain (serine,threonine).

Analogs may be substantially homologous or substantially identical tothe recombinant VWF from which they are derived. Preferred analogs arethose which retain at least some of the biological activity of thewild-type polypeptide, e.g. blood clotting activity.

Polypeptide variants contemplated include polypeptides chemicallymodified by such techniques as ubiquitination, glycosylation, includingpolysialation, conjugation to therapeutic or diagnostic agents,labeling, covalent polymer attachment such as pegylation (derivatizationwith polyethylene glycol), introduction of non-hydrolyzable bonds, andinsertion or substitution by chemical synthesis of amino acids such asornithine, which do not normally occur in human proteins. Variantsretain the same or essentially the same binding properties ofnon-modified molecules of the invention. Such chemical modification mayinclude direct or indirect (e.g., via a linker) attachment of an agentto the VWF polypeptide. In the case of indirect attachment, it iscontemplated that the linker may be hydrolyzable or non-hydrolyzable.

Preparing pegylated polypeptide analogs will generally comprise thesteps of (a) reacting the polypeptide with polyethylene glycol (such asa reactive ester or aldehyde derivative of PEG) under conditions wherebythe binding construct polypeptide becomes attached to one or more PEGgroups, and (b) obtaining the reaction product(s). In general, theoptimal reaction conditions for the acylation reactions will bedetermined based on known parameters and the desired result. Forexample, the larger the ratio of PEG: protein, the greater thepercentage of poly-pegylated product. In some embodiments, the bindingconstruct will have a single PEG moiety at the N-terminus. Polyethyleneglycol (PEG) may be attached to the blood clotting factor to provide alonger half-life in vivo. The PEG group may be of any convenientmolecular weight and may be linear or branched. The average molecularweight of the PEG ranges from about 2 kiloDalton (“kD”) to about 100kDa, from about 5 kDa to about 50 kDa, or from about 5 kDa to about 10kDa. The PEG groups are attached to the blood clotting factor viaacylation or reductive alkylation through a natural or engineeredreactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, orester group) to a reactive group on the blood clotting factor (e.g., analdehyde, amino, or ester group) or by any other technique known in theart.

Methods for preparing polysialylated polypeptide are described in UnitedStates Patent Publication 20060160948, Fernandes et Gregoriadis;Biochim. Biophys. Acta 1341: 26-34, 1997, and Saenko et al., Haemophilia12:42-51, 2006. Briefly, a solution of colominic acid containing 0.1 MNaIO₄ is stirred in the dark at room temperature to oxidize the CA. Theactivated CA solution is dialyzed against, e.g., 0.05 M sodium phosphatebuffer, pH 7.2 in the dark and this solution was added to a rVWFsolution and incubated for 18 h at room temperature in the dark undergentle shaking. Free reagents can then be separated from therVWF-polysialic acid conjugate by ultrafiltration/diafiltration.Conjugation of rVWF with polysialic acid may also be achieved usingglutaraldehyde as cross-linking reagent (Migneault et al., Biotechniques37: 790-796, 2004).

It is further contemplated that a polypeptide of the invention may be afusion protein with a second agent which is a polypeptide. In oneembodiment, the second agent which is a polypeptide, without limitation,is an enzyme, a growth factor, an antibody, a cytokine, a chemokine, acell-surface receptor, the extracellular domain of a cell surfacereceptor, a cell adhesion molecule, or fragment or active domain of aprotein described above. In a related embodiment, the second agent is ablood clotting factor such as Factor VIII, Factor VII, Factor IX. Thefusion protein contemplated is made by chemical or recombinanttechniques well-known in the art.

It is also contemplated that prepro-VWF and pro-VWF polypeptides mayprovide a therapeutic benefit in the formulations of the presentinvention. For example, U.S. Pat. No. 7,005,502 describes apharmaceutical preparation comprising substantial amounts of pro-VWFthat induces thrombin gerneation in vitro. In addition to recombinant,biologically active fragments, variants, or analogs of thenaturally-occurring mature VWF, the present invention contemplates theuse of recombinant biologically active fragments, variants, or analogsof the prepro-VWF (set out in SEQ ID NO:2) or pro-VWF polypeptides(amino acid residues 23 to 764 of SEQ ID NO: 2) in the formulationsdescribed herein.

Polynucleotides encoding fragments, variants and analogs may be readilygenerated by a worker of skill to encode biologically active fragments,variants, or analogs of the naturally-occurring molecule that possessthe same or similar biological activity to the naturally-occurringmolecule. These polynucleotides can be prepared using PCR techniques,digestion/ligation of DNA encoding molecule, and the like. Thus, one ofskill in the art will be able to generate single base changes in the DNAstrand to result in an altered codon and a missense mutation, using anymethod known in the art, including, but not limited to site-specificmutagenesis. As used herein, the phrase “moderately stringenthybridization conditions” means, for example, hybridization at 42° C. in50% formamide and washing at 60° C. in 0.1×SSC, 0.1% SDS. It isunderstood by those of skill in the art that variation in theseconditions occurs based on the length and GC nucleotide base content ofthe sequences to be hybridized. Formulas standard in the art areappropriate for determining exact hybridization conditions. See Sambrooket al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

Formulations and Excipients in General

Excipients are additives that are included in a formulation because theyeither impart or enhance the stability and delivery of a drug product.Regardless of the reason for their inclusion, excipients are an integralcomponent of a drug product and therefore need to be safe and welltolerated by patients. For protein drugs, the choice of excipients isparticularly important because they can affect both efficacy andimmunogenicity of the drug. Hence, protein formulations need to bedeveloped with appropriate selection of excipients that afford suitablestability, safety, and marketability.

The principal challenge in developing formulations for therapeuticproteins is stabilizing the product against the stresses ofmanufacturing, shipping and storage. The role of formulation excipientsis to provide stabilization against these stresses. Excipients may alsobe employed to reduce viscosity of high concentration proteinformulations in order to enable their delivery and enhance patientconvenience. In general, excipients can be classified on the basis ofthe mechanisms by which they stabilize proteins against various chemicaland physical stresses. Some excipients are used to alleviate the effectsof a specific stress or to regulate a particular susceptibility of aspecific protein. Other excipients have more general effects on thephysical and covalent stabilities of proteins. The excipients describedherein are organized either by their chemical type or their functionalrole in formulations. Brief descriptions of the modes of stabilizationare provided when discussing each excipient type.

Given the teachings and guidance provided herein, those skilled in theart will know what amount or range of excipient can be included in anyparticular formulation to achieve a biopharmaceutical formulation of theinvention that promotes retention in stability of the biopharmaceutical(e.g., a polypeptide). For example, the amount and type of a salt to beincluded in a biopharmaceutical formulation of the invention can beselected based on the desired osmolality (i.e., isotonic, hypotonic orhypertonic) of the final solution as well as the amounts and osmolalityof other components to be included in the formulation. Similarly, byexemplification with reference to the type of polyol or sugar includedin a formulation, the amount of such an excipient will depend on itsosmolality.

By way of example, inclusion of about 5% sorbitol can achieveisotonicity while about 9% of a sucrose excipient is needed to achieveisotonicity. Selection of the amount or range of concentrations of oneor more excipients that can be included within a biopharmaceuticalformulation of the invention has been exemplified above by reference tosalts, polyols and sugars. However, those skilled in the art willunderstand that the considerations described herein and furtherexemplified by reference to specific excipients are equally applicableto all types and combinations of excipients including, for example,salts, amino acids, other tonicity agents, surfactants, stabilizers,bulking agents, cryoprotectants, lyoprotectants, anti-oxidants, metalions, chelating agents and/or preservatives.

Further, where a particular excipient is reported in molarconcentration, those skilled in the art will recognize that theequivalent percent (%) w/v (e.g., (grams of substance in a solutionsample/mL of solution)×100%) of solution is also contemplated.

Of course, a person having ordinary skill in the art would recognizethat the concentrations of the excipients described herein share aninterdependency within a particular formulation. By way of example, theconcentration of a bulking agent may be lowered where, e.g., there is ahigh polypeptide concentration or where, e.g., there is a highstabilizing agent concentration. In addition, a person having ordinaryskill in the art would recognize that, in order to maintain theisotonicity of a particular formulation in which there is no bulkingagent, the concentration of a stabilizing agent would be adjustedaccordingly (i.e., a “tonicifying” amount of stabilizer would be used).Common excipients are known in the art and can be found in Powell etal., Compendium of Excipients fir Parenteral Formulations (1998), PDA J.Pharm. Sci. Technology, 52:238-311.

Buffers and Buffering Agents

The stability of a pharmacologically active polypeptide formulation isusually observed to be maximal in a narrow pH range. This pH range ofoptimal stability needs to be identified early during pre-formulationstudies. Several approaches, such as accelerated stability studies andcalorimetric screening studies, have been demonstrated to be useful inthis endeavor (Remmele R. L. Jr., et al., Biochemistry, 38(16): 5241-7(1999)). Once a formulation is finalized, the drug product must bemanufactured and maintained throughout its shelf-life. Hence, bufferingagents are almost always employed to control pH in the formulation.

Organic acids, phosphates and Tris have been employed routinely asbuffers in protein formulations. The buffer capacity of the bufferingspecies is maximal at a pH equal to the pKa and decreases as pHincreases or decreases away from this value. Ninety percent of thebuffering capacity exists within one pH unit of its pKa. Buffer capacityalso increases proportionally with increasing buffer concentration.

Several factors need to be considered when choosing a buffer. First andforemost, the buffer species and its concentration need to be definedbased on its pKa and the desired formulation pH. Equally important is toensure that the buffer is compatible with the polypeptide and otherformulation excipients, and does not catalyze any degradation reactions.A third important aspect to be considered is the sensation of stingingand irritation the buffer may induce upon administration. For example,citrate is known to cause stinging upon injection (Laursen T, et al.,Basic Clin Pharmacol Toxicol., 98(2): 218-21 (2006)). The potential forstinging and irritation is greater for drugs that are administered viathe subcutaneous (SC) or intramuscular (IM) routes, where the drugsolution remains at the site for a relatively longer period of time thanwhen administered by the IV route where the formulation gets dilutedrapidly into the blood upon administration. For formulations that areadministered by direct IV infusion, the total amount of buffer (and anyother formulation component) needs to be monitored. One has to beparticularly careful about potassium ions administered in the form ofthe potassium phosphate buffer, which can induce cardiovascular effectsin a patient (Hollander-Rodriguez J C, et al., Am. Fam. Physician.,73(2): 283-90 (2006)).

The buffer system present in the compositions is selected to bephysiologically compatible and to maintain a desired pH of thepharmaceutical formulation. In one embodiment, the pH of the solution isbetween pH 2.0 and pH 12.0. For example, the pH of the solution may be2.0, 2.3, 2.5, 2.7, 3.0, 3.3, 3.5, 3.7, 4.0, 4.3, 4.5, 4.7, 5.0, 5.3,5.5, 5.7, 6.0, 6.3, 6.5, 6.7, 7.0, 7.3, 7.5, 7.7, 8.0, 8.3, 8.5, 8.7,9.0, 9.3, 9.5, 9.7, 10.0, 10.3, 10.5, 10.7, 11.0, 11.3, 11.5, 11.7, or12.0.

The pH buffering compound may be present in any amount suitable tomaintain the pH of the formulation at a predetermined level. In oneembodiment, the pH buffering concentration is between 0.1 mM and 500 mM(1 M). For example, it is contemplated that the pH buffering agent is atleast 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 500 mM.

Exemplary pH buffering agents used to buffer the formulation as set outherein include, but are not limited to glycine, histidine, glutamate,succinate, phosphate, acetate, citrate, Tris and amino acids or mixturesof amino acids, including, but not limited to aspartate, histidine, andglycine.

Salts

Salts are often added to increase the ionic strength of the formulation,which can be important for protein solubility, physical stability, andisotonicity. Salts can affect the physical stability of proteins in avariety of ways. Ions can stabilize the native state of proteins bybinding to charged residues on the protein's surface. Alternatively,salts can stabilize the denatured state by binding to peptide groupsalong the protein backbone (—CONH—). Salts can also stabilize theprotein native conformation by shielding repulsive electrostaticinteractions between residues within a protein molecule. Salts inprotein formulations can also shield attractive electrostaticinteractions between protein molecules that can lead to proteinaggregation and insolubility. In formulations provided, the saltconcentration is between 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150,200, 300, and 500 mM.

Stabilizers and Bulking Agents

In the present pharmaceutical formulations, a stabilizer (or acombination of stabilizers) may be added to prevent or reducestorage-induced aggregation and chemical degradation. A hazy or turbidsolution upon reconstitution indicates that the protein has precipitatedor at least aggregated. The term “stabilizer” means an excipient capableof preventing aggregation or other physical degradation, as well aschemical degradation (for example, autolysis, deamidation, oxidation,etc.) in an aqueous state. Stabilizers that are conventionally employedin pharmaceutical compositions include, but are not limited to, sucrose,trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose,gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol,sorbitol, glycine, arginine HCL, poly-hydroxy compounds, includingpolysaccharides such as dextran, starch, hydroxyethyl starch,cyclodextrins, N-methylpyrollidene, cellulose and hyaluronic acid,sodium chloride, [Carpenter et al., Develop. Biol. Standard 74:225,(1991)]. In the present formulations, the stabilizer is incorporated ina concentration of about 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM.

If desired, the formulations also include appropriate amounts of bulkingand osmolarity regulating agents. Bulking agents include, for example,mannitol, glycine, sucrose, polymers such as dextran,polyvinylpyrolidone, carboxymethylcellulose, lactose, sorbitol,trehalose, or xylitol. In one embodiment, the bulking agent is mannitol.The bulking agent is incorporated in a concentration of about 0.1, 0.5,0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,500, 700, 900, or 1000 mM.

Surfactants

Protein molecules have a high propensity to interact with surfacesmaking them susceptible to adsorption and denaturation at air-liquid,vial-liquid, and liquid-liquid (silicone oil) interfaces. Thisdegradation pathway has been observed to be inversely dependent onprotein concentration and results in either the formation of soluble andinsoluble protein aggregates or the loss of protein from solution viaadsorption to surfaces. In addition to container surface adsorption,surface-induced degradation is exacerbated with physical agitation, aswould be experienced during shipping and handling of the product.

Surfactants are commonly used in protein formulations to preventsurface-induced degradation. Surfactants are amphipathic molecules withthe capability of out-competing proteins for interfacial positions.Hydrophobic portions of the surfactant molecules occupy interfacialpositions (e.g., air/liquid), while hydrophilic portions of themolecules remain oriented towards the bulk solvent. At sufficientconcentrations (typically around the detergent's critical micellarconcentration), a surface layer of surfactant molecules serve to preventprotein molecules from adsorbing at the interface. Thereby,surface-induced degradation is minimized. The most commonly usedsurfactants are fatty acid esters of sorbitan polyethoxylates, i.e.polysorbate 20 and polysorbate 80. The two differ only in the length ofthe aliphatic chain that imparts hydrophobic character to the molecules,C-12 and C-18, respectively. Accordingly, polysorbate-80 is moresurface-active and has a lower critical micellar concentration thanpolysorbate-20.

Detergents can also affect the thermodynamic conformational stability ofproteins. Here again, the effects of a given detergent excipient will beprotein specific. For example, polysorbates have been shown to reducethe stability of some proteins and increase the stability of others.Detergent destabilization of proteins can be rationalized in terms ofthe hydrophobic tails of the detergent molecules that can engage inspecific binding with partially or wholly unfolded protein states. Thesetypes of interactions could cause a shift in the conformationalequilibrium towards the more expanded protein states (i.e. increasingthe exposure of hydrophobic portions of the protein molecule incomplement to binding polysorbate). Alternatively, if the protein nativestate exhibits some hydrophobic surfaces, detergent binding to thenative state may stabilize that conformation.

Another aspect of polysorbates is that they are inherently susceptibleto oxidative degradation. Often, as raw materials, they containsufficient quantities of peroxides to cause oxidation of protein residueside-chains, especially methionine. The potential for oxidative damagearising from the addition of stabilizer emphasizes the point that thelowest effective concentrations of excipients should be used informulations. For surfactants, the effective concentration for a givenprotein will depend on the mechanism of stabilization. It has beenpostulated that if the mechanism of surfactant stabilization is relatedto preventing surface-denaturation the effective concentration will bearound the detergent's critical micellar concentration. Conversely, ifthe mechanism of stabilization is associated with specificprotein-detergent interactions, the effective surfactant concentrationwill be related to the protein concentration and the stoichiometry ofthe interaction (Randolph T. W., et al., Pharm Biotechnol., 13:159-75(2002)).

Surfactants may also be added in appropriate amounts to prevent surfacerelated aggregation phenomenon during freezing and drying [Chang, B, J.Pharm. Sci. 85:1325, (1996)]. Exemplary surfactants include anionic,cationic, nonionic, zwitterionic, and amphoteric surfactants includingsurfactants derived from naturally-occurring amino acids. Anionicsurfactants include, but are not limited to, sodium lauryl sulfate,dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate,chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecylsulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate,sodium deoxycholate, and glycodeoxycholic acid sodium salt. Cationicsurfactants include, but are not limited to, benzalkonium chloride orbenzethonium chloride, cetylpyridinium chloride monohydrate, andhexadecyltrimethylammonium bromide. Zwitterionic surfactants include,but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Non-ionicsurfactants include, but are not limited to, digitonin, Triton X-100,Triton X-114, TWEEN-20, and TWEEN-80. Surfactants also include, but arenot limited to lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylenehydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate,polysorbate 40, 60, 65 and 80, soy lecithin and other phospholipids suchas dioleyl phosphatidyl choline (DOPC), dimyristoylphosphatidyl glycerol(DMPG), dimyristoylphosphatidyl choline (DMPC), and (dioleylphosphatidyl glycerol) DOPG; sucrose fatty acid ester, methyl celluloseand carboxymethyl cellulose. Compositions comprising these surfactants,either individually or as a mixture in different ratios, are thereforefurther provided. In the present formulations, the surfactant isincorporated in a concentration of about 0.01 to about 0.5 g/L.

Other Common Excipient Components

Amino Acids

Amino acids have found versatile use in protein formulations as buffers,bulking agents, stabilizers and antioxidants. Histidine and glutamicacid are employed to buffer protein formulations in the pH range of5.5-6.5 and 4.0-5.5 respectively. The imidazole group of histidine has apKa=6.0 and the carboxyl group of glutamic acid side chain has a pKa of4.3 which makes these amino acids suitable for buffering in theirrespective pH ranges. Glutamic acid is particularly useful in such cases(e.g., Stemgen®). Histidine is commonly found in marketed proteinformulations (e.g., Xolair®, Herceptin®, Recombinate®), and this aminoacid provides an alternative to citrate, a buffer known to sting uponinjection. Interestingly, histidine has also been reported to have astabilizing effect, as observed in formulations with ABX-IL8 (an IgG2antibody), with respect to aggregation when used at high concentrationsin both liquid and lyophilized presentations (Chen B, et al., PharmRes., 20(12): 1952-60 (2003)). Histidine (up to 60 mM) was also observedto reduce the viscosity of a high concentration formulation of thisantibody. However, in the same study, the authors observed increasedaggregation and discoloration in histidine containing formulationsduring freeze-thaw studies of the antibody in stainless steelcontainers. The authors attributed this to an effect of iron ionsleached from corrosion of steel containers. Another note of caution withhistidine is that it undergoes photo-oxidation in the presence of metalions (Tomita M, et al., Biochemistry, 8(12): 5149-60 (1969)). The use ofmethionine as an antioxidant in formulations appears promising; it hasbeen observed to be effective against a number of oxidative stresses(Lam X M, et al., J Pharm Sci., 86(11): 1250-5 (1997)).

The amino acids glycine, proline, serine and alanine have been shown tostabilize proteins by the mechanism of preferential exclusion. Glycineis also a commonly used bulking agent in lyophilized formulations (e.g.,Neumega®, Genotropin®, Humatrope®). Arginine has been shown to be aneffective agent in inhibiting aggregation and has been used in bothliquid and lyophilized formulations (e.g., Activase®, Avonex®, Enbrel®liquid). Furthermore, the enhanced efficiency of refolding of certainproteins in the presence of arginine has been attributed to itssuppression of the competing aggregation reaction during refolding.

Antioxidants

Oxidation of protein residues arises from a number of different sources.Beyond the addition of specific antioxidants, the prevention ofoxidative protein damage involves the careful control of a number offactors throughout the manufacturing process and storage of the productsuch as atmospheric oxygen, temperature, light exposure, and chemicalcontamination. The most commonly used pharmaceutical antioxidants arereducing agents, oxygen/free-radical scavengers, or chelating agents.Antioxidants in therapeutic protein formulations are water-soluble andremain active throughout the product shelf-life. Reducing agents andoxygen/free-radical scavengers work by ablating active oxygen species insolution. Chelating agents such as EDTA are effective by binding tracemetal contaminants that promote free-radical formation. For example,EDTA was utilized in the liquid formulation of acidic fibroblast growthfactor to inhibit the metal ion catalyzed oxidation of cysteineresidues. EDTA has been used in marketed products like Kineret® andOntak®.

In addition to the effectiveness of various excipients to preventprotein oxidation, the potential for the antioxidants themselves toinduce other covalent or physical changes to the protein is of concern.For example, reducing agents can cause disruption of intramoleculardisulfide linkages, which can lead to disulfide shuffling. In thepresence of transition metal ions, ascorbic acid and EDTA have beenshown to promote methionine oxidation in a number of proteins andpeptides (Akers M J, and Defelippis M R. Peptides and Proteins asParenteral Solutions. In: Pharmaceutical Formulation Development ofPeptides and Proteins. Sven Frokjaer, Lars Hovgaard, editors.Pharmaceutical Science. Taylor and Francis, UK (1999)); Fransson J. R.,J. Pharm. Sci. 86(9): 4046-1050 (1997); Yin J, et al., Pharm Res.,21(12): 2377-83 (2004)). Sodium thiosulfate has been reported to reducethe levels of light and temperature induced methionine-oxidation inrhuMab HER2; however, the formation of a thiosulfate-protein adduct wasalso reported in this study (Lam X M, Yang J Y, et al., J Pharm Sci.86(11): 1250-5 (1997)). Selection of an appropriate antioxidant is madeaccording to the specific stresses and sensitivities of the protein.

Metal Ions

In general, transition metal ions are undesired in protein formulationsbecause they can catalyze physical and chemical degradation reactions inproteins. However, specific metal ions are included in formulations whenthey are co-factors to proteins and in suspension formulations ofproteins where they form coordination complexes (e.g., zinc suspensionof insulin). Recently, the use of magnesium ions (10-120 mM) has beenproposed to inhibit the isomerization of aspartic acid to isoasparticacid (WO 2004039337).

Two examples where metal ions confer stability or increased activity inproteins are human deoxyribonuclease (rhDNase, Pulmozyme®), and FactorVIII. In the case of rhDNase, Ca⁺² ions (up to 100 mM) increased thestability of the enzyme through a specific binding site (Chen B, et al.,J Pharm Sci., 88(4): 477-82 (1999)). In fact, removal of calcium ionsfrom the solution with EGTA caused an increase in deamidation andaggregation. However, this effect was observed only with Ca⁺² ions;other divalent cations Mg⁺², Mn⁺² and Zn⁺² were observed to destabilizerhDNase. Similar effects were observed in Factor VIII. Ca⁺² and Sr⁺²ions stabilized the protein while others like Mg⁺², Mn⁺² and Zn⁺², Cu⁺²and Fe⁺² destabilized the enzyme (Fatouros, A., et al., Int. J. Pharm.,155, 121-131 (1997). In a separate study with Factor VIII, a significantincrease in aggregation rate was observed in the presence of Al⁺³ ions(Derrick T S, et al., J. Pharm. Sci., 93(10): 2549-57 (2004)). Theauthors note that other excipients like buffer salts are oftencontaminated with Al⁺³ ions and illustrate the need to use excipients ofappropriate quality in formulated products.

Preservatives

Preservatives are necessary when developing multi-use parenteralformulations that involve more than one extraction from the samecontainer. Their primary function is to inhibit microbial growth andensure product sterility throughout the shelf-life or term of use of thedrug product. Commonly used preservatives include benzyl alcohol, phenoland m-cresol. Although preservatives have a long history of use, thedevelopment of protein formulations that includes preservatives can bechallenging. Preservatives almost always have a destabilizing effect(aggregation) on proteins, and this has become a major factor inlimiting their use in multi-dose protein formulations (Roy S, et al., JPharm Sci., 94(2): 382-96 (2005)).

To date, most protein drugs have been formulated for single-use only.However, when multi-dose formulations are possible, they have the addedadvantage of enabling patient convenience, and increased marketability.A good example is that of human growth hormone (hGH) where thedevelopment of preserved formulations has led to commercialization ofmore convenient, multi-use injection pen presentations. At least foursuch pen devices containing preserved formulations of hGH are currentlyavailable on the market. Norditropin® (liquid, Novo Nordisk), NutropinAQ® (liquid, Genentech) & Genotropin (lyophilized—dual chambercartridge, Pharmacia & Upjohn) contain phenol while Somatrope® (EliLilly) is formulated with m-cresol.

Several aspects need to be considered during the formulation developmentof preserved dosage forms. The effective preservative concentration inthe drug product must be optimized. This requires testing a givenpreservative in the dosage form with concentration ranges that conferanti-microbial effectiveness without compromising protein stability. Forexample, three preservatives were successfully screened in thedevelopment of a liquid formulation for interleukin-1 receptor (Type I),using differential scanning calorimetry (DSC). The preservatives wererank ordered based on their impact on stability at concentrationscommonly used in marketed products (Remmele R L Jr., et al., Pharm Res.,15(2): 200-8 (1998)).

Some preservatives can cause injection site reactions, which is anotherfactor that needs consideration when choosing a preservative. Inclinical trials that focused on the evaluation of preservatives andbuffers in Norditropin, pain perception was observed to be lower informulations containing phenol and benzyl alcohol as compared to aformulation containing m-cresol (Kappelgaard A. M., Horm Res. 62 Suppl3:98-103 (2004)). Interestingly, among the commonly used preservative,benzyl alcohol possesses anesthetic properties (Minogue S C, and Sun DA., Anesth Analg., 100(3): 683-6 (2005)).

Lyophilization

It is also contemplated that the formulations comprising a VWFpolypeptide of the invention may be lyophilized prior to administration.Lyophilization is carried out using techniques common in the art andshould be optimized for the composition being developed [Tang et al.,Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9(1996)].

A lyophilization cycle is, in one aspect, composed of three steps:freezing, primary drying, and secondary drying [A. P. Mackenzie, PhilTrans R Soc London, Ser B, Biol 278:167 (1977)]. In the freezing step,the solution is cooled to initiate ice formation. Furthermore, this stepinduces the crystallization of the bulking agent. The ice sublimes inthe primary drying stage, which is conducted by reducing chamberpressure below the vapor pressure of the ice, using a vacuum andintroducing heat to promote sublimation. Finally, adsorbed or boundwater is removed at the secondary drying stage under reduced chamberpressure and at an elevated shelf temperature. The process produces amaterial known as a lyophilized cake. Thereafter the cake can bereconstituted with either sterile water or suitable diluent forinjection.

The lyophilization cycle not only determines the final physical state ofthe excipients but also affects other parameters such as reconstitutiontime, appearance, stability and final moisture content. The compositionstructure in the frozen state proceeds through several transitions(e.g., glass transitions, wettings, and crystallizations) that occur atspecific temperatures and can be used to understand and optimize thelyophilization process. The glass transition temperature (Tg and/or Tg′)can provide information about the physical state of a solute and can bedetermined by differential scanning calorimetry (DSC). Tg and Tg′ are animportant parameter that must be taken into account when designing thelyophilization cycle. For example, Tg′ is important for primary drying.Furthermore, in the dried state, the glass transition temperatureprovides information on the storage temperature of the final product.

Methods of Preparation

The present invention further contemplates methods for the preparationof pharmaceutical formulations. A variety of aqueous carriers, e.g.,sterile water for injection, water with preservatives for multi doseuse, or water with appropriate amounts of surfactants (for example,polysorbate-20), 0.4% saline, 0.3% glycine, or aqueous suspensions maycontain the active compound in admixture with excipients suitable forthe manufacture of aqueous suspensions. In various aspects, suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate.

Administration

To administer compositions to human or test animals, in one aspect, thecompositions comprises one or more pharmaceutically acceptable carriers.The phrases “pharmaceutically” or “pharmacologically” acceptable referto molecular entities and compositions that are stable, inhibit proteindegradation such as aggregation and cleavage products, and in additiondo not produce allergic, or other adverse reactions when administeredusing routes well-known in the art, as described below.“Pharmaceutically acceptable carriers” include any and all clinicallyuseful solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like,including those agents disclosed above.

The pharmaceutical formulations may be administered orally, topically,transdermally, parenterally, by inhalation spray, vaginally, rectally,or by intracranial injection. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intracisternal injection, or infusion techniques. Administration byintravenous, intradermal, intramuscular, intramammary, intraperitoneal,intrathecal, retrobulbar, intrapulmonary injection and or surgicalimplantation at a particular site is contemplated as well. Generally,compositions are essentially free of pyrogens, as well as otherimpurities that could be harmful to the recipient.

Single or multiple administrations of the compositions can be carriedout with the dose levels and pattern being selected by the treatingphysician. For the prevention or treatment of disease, the appropriatedosage will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether drug isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the drug, and thediscretion of the attending physician.

Kits

As an additional aspect, the invention includes kits which comprise oneor more pharmaceutical formulations packaged in a manner whichfacilitates their use for administration to subjects. In one embodiment,such a kit includes pharmaceutical formulation described herein (e.g., acomposition comprising a therapeutic protein or peptide), packaged in acontainer such as a sealed bottle or vessel, with a label affixed to thecontainer or included in the package that describes use of the compoundor composition in practicing the method. In one embodiment, thepharmaceutical formulation is packaged in the container such that theamount of headspace in the container (e.g., the amount of air betweenthe liquid formulation and the top of the container) is very small.Preferably, the amount of headspace is negligible (i.e., almost none).In one embodiment, the kit contains a first container having atherapeutic protein or peptide composition and a second container havinga physiologically acceptable reconstitution solution for thecomposition. In one aspect, the pharmaceutical formulation is packagedin a unit dosage form. The kit may further include a device suitable foradministering the pharmaceutical formulation according to a specificroute of administration. Preferably, the kit contains a label thatdescribes use of the pharmaceutical formulations.

Dosages

The dosage regimen involved in a method for treating a conditiondescribed herein will be determined by the attending physician,considering various factors which modify the action of drugs, e.g. theage, condition, body weight, sex and diet of the patient, the severityof any infection, time of administration and other clinical factors. Byway of example, a typical dose of a recombinant VWF of the presentinvention is approximately 50 U/kg, equal to 500 μg/kg.

Formulations of the invention may be administered by an initial bolusfollowed by a continuous infusion to maintain therapeutic circulatinglevels of drug product. As another example, the inventive compound maybe administered as a one-time dose. Those of ordinary skill in the artwill readily optimize effective dosages and administration regimens asdetermined by good medical practice and the clinical condition of theindividual patient. The frequency of dosing will depend on thepharmacokinetic parameters of the agents and the route ofadministration. The optimal pharmaceutical formulation will bedetermined by one skilled in the art depending upon the route ofadministration and desired dosage. See for example, Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712, the disclosure of which is herebyincorporated by reference. Such formulations may influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the administered agents. Depending on the route of administration, asuitable dose may be calculated according to body weight, body surfacearea or organ size. Appropriate dosages may be ascertained through useof established assays for determining blood level dosages in conjunctionwith appropriate dose-response data. The final dosage regimen will bedetermined by the attending physician, considering various factors whichmodify the action of drugs, e.g. the drug's specific activity, theseverity of the damage and the responsiveness of the patient, the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. As studiesare conducted, further information will emerge regarding the appropriatedosage levels and duration of treatment for various diseases andconditions.

The following examples are not intended to be limiting but onlyexemplary of specific embodiments of the invention.

Example 1 Shaking Experiments

In order to determine the amount of precipitation of rVWF in variousformulations, the percent recovery of rVWF following turbulent shakingwas tested under a variety of conditions.

rVWF in Advate buffer (90 mM NaCl, 1.68 mM CaCl₂, 10 mM L-histidine, 1mM tris, 0.26 mM glutathione, 23.4 mM trehalose, 175.7 mM mannitol, and0.1 g/L TWEEN-80, pH 7.0) or Advate 1:3 buffer (Advate buffer diluted3-fold in water) was subjected to turbulent shaking on a shaker at roomtemperature (RT) for 0 min, 1 min, 2.5 hrs, or 4 days, and percentrecovery of the rVWF was measured relative to the starting materialprior to shaking. As shown in Table 1, losses of about 40-80% wereobserved in the Advate buffer while losses of about 20-30% were observedin the Advate 1:3 buffer. VWF antigen VWF:Ag corresponds to the amountof VWF which can be detected in an VWF-specific ELISA using polyclonalanti-VWF antibody, while VWF:RCo corresponds to the amount of VWF whichcauses agglutination of stabilized platelets in the presence ofristocetin. In both cases human reference plasma calibrated against theactual WHO standard was used as standard (1 ml of reference plasmausually contains 1U VWF).

TABLE 1 Influence of turbulent shaking time on rVWF recovery Turbulentshaking at VWF:Ag Recovery VWF:RCo Recovery RCo/VWF:Ag rVWF RT [U/ml][%] [U/ml] [%] [U/U] Advate 0 min 213 100%  104 100% 0.49 1 min 120 56%2.5 hr 139 65% 4 d 37 17% 7  7% 0.19 Advate 0 min 206 100%  134 100%0.65 1:3 1 min 152 74% 2.5 hr 170 82% 4 d 138 67% 131  98% 0.95

The effect of freeze/thawing and lyophilization was also tested in theshaking experiments. Freezing was performed at −20° C. in an −20° C.cold room or on dry ice, thawing in both cases at RT and both startedfrom the liquid formulations. As for lyophilization, the formulated VWFsamples described herein were frozen within a pilot scale lyophilizer at<=−40° C. and were lyophilized using a standard Iyo program. Shaking wasperformed directly with the liquid formulations (2 ml in 5 ml vials). Asshown in Table 2, percent recovery of rVWF was higher in Advate 1:3buffer compared to Advate buffer.

TABLE 2 VWF:Ag VWF:RCo VWF:Ag recovery VWF:RCo recovery RCo:Ag RVWF[U/ml] [%] [U/ml] [%] [U/U] Advate Frozen 213 100% 104 100%  0.49Frozen - 3x 229 107% 84 81% 0.37 at −20° C. Frozen - 3x 231 108% 72 69%0.31 with dry ice Lyo 242 113% 61 59% 0.25 Starting 213 100% 104 100% 0.49 material Heavily 37.0  17% 7.2 6.9%  0.19 shaken for 4 days at RTAdvate Frozen 206 100% 134 100%  0.65 1:3 Frozen - 3x 184  89% 132 99%0.72 at −20° C. Frozen - 3x 195  94% 128 96% 0.66 with dry ice Lyo 195 94% 107 80% 0.55 Starting 206 100% 134 100%  0.65 material Heavily 138 67% 131 98% 0.95 shaken for 4 days at RT

Percent recovery was also measured in the shaking experiments with rVWFbeing stored in syringes with headspace and without headspace.Interestingly, when rVWF is stored in syringes without headspace andshaken as described above, no rVWF precipitation was observed. Incontrast, when rVWF is stored in syringes with headspace, someprecipitation was observed.

In summary, turbulent shaking resulted in at least 30% loss of rVWF inAdvate buffer or Advate 1:3 buffer, with Advate buffer showing higherloss of recovery compared to Advate 1:3 buffer. Interestingly, the sameprecipitates observed in the turbulent shaking experiments were notobserved when rVWF was stored and transported ˜5000 km in an automobile(representing the expected shaking during transport). Precipitation ofrVWF could be eliminated by storage in syringes without headspace.

Example 2 Stability of Recombinant VWF

The stability of rVWF was tested by assessing the activity level of rVWFpresent in a various formulations.

As shown in FIG. 1, rVWF is not stable in Advate buffer after 26 weeksdue to the presence of 0.3 mM glutathione. As shown in FIG. 2, however,rVWF is more stable in Advate 1:3 buffer (e.g., for up 12 weeks at 4°C.)

As shown in FIG. 3, the stability of a citrate-based formulation (15 mMsodium citrate, 10 mM CaCl₂, 100 mM NaCl, pH 7.0) is better than Advate1:3 buffer formulation containing 0.1M glutathione.

Likewise, the concentration of rVWF was measured over time in variousbuffers. As shown in FIG. 4, FIG. 5 and FIG. 6, rVWF concentration isstable over time in Advate buffer, Advate 1:3 buffer, and citrate-basedbuffer, respectively.

Example 4 Characterization of the Liquid Formulations

Differential scanning calorimetry (DSC) was used to assess the extent ofprotein (rVWF) unfolding in various buffers. As shown in Table 3, Advatebuffer pH 7.0 is the optimum for stabilization.

DSC is a thermoanalytical technique in which the difference in theamount of heat required to increase the temperature of a sample andreferences are measured as a function of temperature. The result of aDSC experiment is a curve of heat flux versus temperature or versustime.

The Differential Scanning Calorimeter can scan through a range oftemperatures while heating and cooling and it determines a phasetransition, i.e. melting, crystallization, or glass transition, bymeasuring the amount of heat needed to reach a set temperature. Thecalorimeter was calibrated with a set of pure metals (zinc, indium, andtin) that have a known heat capacity, Cp and melting point, Tm. Therespective reference buffer was placed into the reference capillary andthe rVWF sample was placed into the sample capillary of the instrument.

TABLE 3 Unfolding temperature in various buffers Lot Buffer pH T unfold[° C] rVWF161A Advate 7.0 66.0 rVWF161B Immunate 6.8 64.5 rVWF161CCitrate 6.8 61.2 rVWF161D NovoSeven 6.8 64.9 rVWF158 Hepes 7.4 61.3Buffer components and concentrations:

A) Advate: 5.26 g/l NaCl pH = 7.0 0.248 g/l CaCl2 32 g/l D-Mannitol 8g/l Trehalose 1.56 g/l L-Histidine 1.2 g/l Tris 0.08 g/l Glutadione red.B) Immunate: 5.25 g/l Glycin pH = 6.8 2.2 g/l NaCl 5.25 g/l NaCit3 5.25g/l Lysin-HCl 0.62 g/l CaCl2 C) Citrat: 3 g/l Glycin pH = 6.8 2.92 g/lNaCl 2.5 g/l NaCit3 30 g/l D-Mannitol 10 g/l Trehalose D) NovoSeven:0.75 g/l Glycin pH = 6.8 2.92 g/l NaCl 1.47 g/l CaCl2 30 g/l D-Mannitol

rVWF158: 20 mM Hepes, 150 mM NaCl, 5 g/L sucrose, pH 7.4

Further, as shown in FIG. 7, most formulation excipients increase theunfolding temperature by about 1-2° C. FIG. 8 shows that 10 mM CaCl₂increases the unfolding temperature by ˜8° C. to ˜67° C., an unfoldingtemperature which can also be reached by Advate buffer. This effect ofCaCl₂ is similar at pH 7.3 and 6.5, as shown in FIG. 9. Finally, theeffect of trehalose and sucrose were analyzed on the unfoldingtemperature. Compared to citrate alone, neither trehalose nor sucroseincreased the unfolding temperature of rVWF. A summary of the unfoldingtemperature (Tmax) data for rVWF in the presence of various excipientsis set out in Table 4.

TABLE 4 15 mM Sodium 15 mM 50 mM Citrate buffer — 15 mM Tris GlycineNaCl ΔH [kJ/mol] 128494.3 656259.7 157352.2 124985.8 Unfolding T 58.659.1 61 [° C.] - Peak 1 Peak 2 65.2 68.5 65.5 Peak 3 80.4 80.1 81 Peak 415 mM Sodium 15 mM 20.52 g/L 10.26 g/L Citrate buffer Histidine MannitolTrehalose ΔH [kJ/mol] 134044.5 1588590.1 612235.9 Unfolding T 59.2 58.558.5 [° C.] - Peak 1 Peak 2 65.2 65.5 71.3 Peak 3 79.3 78.2 81.5 Peak 488.5 92.7 0.25 mM 15 mM Sodium 32 g/L Sac- Citrate buffer 1 mM CaCl₂ 10mM CaCl₂ Saccharose charose ΔH [kJ/mol] 266008.2 308171.3 115082.4246904.6 Unfolding T 64.5 67.2 59.2 60 [° C.] - Peak 1 Peak 2 66 67 Peak3 81 83.1 81.1 81.7 Peak 4 91.8 93 32 g/L 15 mM Sodium 0.1 g/L Raf-Na₂HPO₄/ 7.8 mM Citrate buffer TWEEN-80 finose NaHPO₄ Trehalose ΔH[kJ/mol] 338792.7 127329.2 197967.5 135573.3 Unfolding T 58.7 60.1 61.458.4 [° C.] - Peak 1 Peak 2 64.4 65.8 65.4 Peak 3 81.6 80.3 80.4 80.4Peak 4 89.2

In addition to the various buffers, DSC was used to assess unfoldingtemperature of rVWF at various pH values in Advate buffer. The resultsare shown in Table 5, below. Advate buffer pH 7.0 is the optimum forstabilization (i.e., highest unfolding temperature; Peak 1) of rVWF.

TABLE 5 pH Peak 1 Peak 2 5.0 59.5 62.0 6.0 65.2 75.4 7.0 67.2 82.8 8.066.6 85.6 9.0 65.0 84.9

The fluorescence spectrum of rVWF in Advate buffer and Advate 1:3 bufferwas assessed after storage at various temperatures for various lengthsof time. No (or only slight) change in fluorescence spectrum wasobserved after storage at 40° C. from 0 to 28 days in either Advate orAdvate 1:3 buffers. No difference was observed at other temperatures.

Likewise, degradation of rVWF was assessed using gelfiltration (Superose6). While some degradation was observed after 26 weeks at 4° C. inAdvate buffer, almost no degradation of rVWF in Advate 1:3 buffer wasobserved after 26 weeks at 4° C. At 40° C., glutathione increased theamount of degradation over time (albeit to a slower extent in Advate 1:3buffer).

Based on the above Examples, Advate 1:3 buffer offers an advantage withrespect to freeze/thawing and recovery after lyophilization as comparedto the undiluted Advate buffer. Moreover, Advate 1:3 buffer canstabilize (e.g., maintain biological activity) rVWF activity duringincubation at 40° C. better that Advate buffer. rVWF in Advate 1:3buffer is stable for 4 weeks of incubation at 4° C. Finally, DSC hasdemonstrated that pH 7.0 is optimum for preventing degradation of rVWF(i.e., showed the highest unfolding temperature).

Thus, in view of the data presented herein, a formulation was proposedfor rVWF including 15 mM citrate (or glycine or hitidine), 10 mM CaCl₂,pH 6.5-7.3, adjusted to the desired osmolarity by NaCl. For example, inone embodiment, the citrate-based formula is 15 mM sodium citrate, 10 mMCaCl₂, 100 mM NaCl, pH 7.0.

Alternatively, an Advate or Advate 1:3 buffer, without glutathione, isalso contemplated: Advate: 90 mM NaCl, 1.68 mM CaCl₂, 10 mM L-histidine,10 mM Tris, 0.26 mM glutathione, 23.4 mM trehalose, 175.7 mM mannitol,and 0.1 g/L TWEEN-80, pH 7.0; Advate 1:3: 30 mM NaCl, 0.56 mM CaCl₂, 3.3mM L-histidine, 3.3 mM tris, 7.8 mM trehalose, 58.6 mM mannitol, and0.03 g/L TWEEN-80, ph 7.0.

1. A stable liquid pharmaceutical formulation of a recombinant vonWillebrand Factor (rVWF) comprising: (a) a rVWF; (b) a buffering agent;(c) one or more salts; (d) optionally a stabilizing agent; and (e)optionally a surfactant; wherein said rVWF comprises a polypeptideselected from the group consisting of: a) the amino acid sequence setout in SEQ ID NO: 3; b) a biologically active analog, fragment orvariant of a); c) a polypeptide encoded by the polynucleotide set out inSEQ ID NO: 1; d) a biologically active analog, fragment or variant ofc); and e) a polypeptide encoded by a polynucleotide that hybridizes tothe polynucleotide set out in SEQ ID NO: 1 under moderately stringenthybridization conditions; wherein said buffer is comprised of a pHbuffering agent in a range of about 0.1 mM to about 500 mM and whereinthe pH is in a range of about 2.0 to about 12.0; wherein said salt is ata concentration of about 1 to 500 mM; wherein said stabilizing agent isat a concentration of about 0.1 to 1000 mM; and wherein said surfactantis at a concentration of about 0.01 g/L to 0.5 g/L.
 2. The formulationof claim 1 wherein the rVWF comprises the amino acid sequence set out inSEQ ID NO:
 3. 3. The formulation of claim 1 wherein the buffering agentis selected from the group consisting of sodium citrate, glycine,histidine, Tris and combinations of these agents.
 4. The formulation ofclaim 3 wherein the buffering agent is sodium citrate at a concentrationof 15 mM.
 5. The formulation of claim 1 wherein pH is in the range of6.0-8.0.
 6. The formulation of claim 5 wherein pH is in the range of6.5-7.3.
 7. The formulation of claim 4 wherein the pH is 7.0.
 8. Theformulation of claim 1 wherein the buffering agent is citrate and the pHis 7.0.
 9. The formulation of claim 1 wherein the salt is selected fromthe group consisting of calcium chloride, sodium chloride and magnesiumchloride.
 10. The formulation of claim 9 wherein the salt is at aconcentration range of 0.5 to 300 mM.
 11. The formulation of claim 10wherein the salt is calcium chloride at a concentration of 10 mM. 12.The formulation of claim 1 wherein the rVWF comprises the amino acidsequence set out in SEQ ID NO: 3; wherein the buffering agent is citrateand the pH is 7.0; and wherein the salt is calcium chloride at aconcentration of 10 mM.
 13. The formulation of claim 1 wherein the rVWFcomprises the amino acid sequence set out in SEQ ID NO: 3; wherein thebuffering agent is sodium citrate at a concentration of 15 mM and the pHis 7.0; and wherein the salt is calcium chloride at a concentration of10 mM and NaCl at a concentration of 100 mM.
 14. The formulation ofclaim 3 wherein the one or more buffering agents is histidine and Trisat a concentration of 3.3 mM each.
 15. The formulation of claim 3wherein the pH is 7.0.
 16. The formulation of claim 9 wherein the one ormore salts is sodium chloride at a concentration of 30 mM and calciumchloride at a concentration of 0.56 mM.
 17. The formulation of claim 1wherein the stabilizing agent is selected from the group consisting ofmannitol, lactose, sorbitol, xylitol, sucrose, trehalose, mannose,maltose, lactose, glucose, raffinose, cellobiose, gentiobiose,isomaltose, arabinose, glucosamine, fructose and combinations of thesestabilizing agents.
 18. The formulation of claim 17 wherein thestabilizing agents are trehalose at a concentration of 7.8 mM andmannitol at a concentration of 58.6 mM.
 19. The formulation of claim 1wherein the surfactant is selected from the group consisting ofdigitonin, Triton X-100, Triton X-114, TWEEN-20, TWEEN-80 andcombinations of these surfactants.
 20. The formulation of claim 1wherein the surfactant is TWEEN-80 at 0.03 g/L.
 21. The formulation ofclaim 1 wherein the rVWF comprises amino acid sequence set out in SEQ IDNO: 3; wherein the buffering agents are histidine at a concentration of3.3 mM and Tris at a concentration of 3.3 mM at pH 7.0; wherein thesalts are sodium chloride at a concentration of 30 mM and calciumchloride at a concentration of 0.56 mM; wherein the stabilizing agentsare trehalose at a concentration of 7.8 mM and mannitol at aconcentration of 58.6 mM.; and wherein the surfactant is TWEEN-80 at0.03 g/L.